The invention relates to methods of making antibodies. More specifically, the invention relates to the selective production of monoclonal antibodies having predefined specificity.
Almost a century ago, the discovery of ABO blood groups by Karl Landsteiner (1901) led to the development of routine blood grouping procedures in the practice of pretransfusion testing performed today. This testing depends on agglutination resulting from the interaction between the red blood cells (RBCs) and their cognate antibodies. Historically, human-source reagents have been used for blood group antigen typing. From time to time attempts have been made to use non-human reagents for blood grouping. Injection of human RBCs to animals, such as mice and rabbits, often results in production of a mixture of antibodies which require extensive absorption or purification for use to detect a single polymorphic determinant. However, with the advent of the ability to produce monoclonal antibodies (MAbs) having predefined specificities as described in the mid-seventies (Kohler et al. 1975), and in view of the legally restricted immunization of individuals with allogeneic RBCs, a new era began in the blood typing industry.
In producing these new monoclonal antibodies, after immunization of mice with appropriate antigens (e.g., RBCs), an antibody-secreting spleen cell is isolated and fused with an "immortal cell" (a myeloma cell line), to create a hybrid cell called a "hybridoma." The resulting hybridoma uniquely secretes the antibody of interest into the culture medium, but is also immortal, thereby capable of acting as a continuous source of the antibody.
The first murine MAb-based blood typing reagent, an anti-M, was licensed by the Food and Drug Administration in 1984 and since that date, MAb reagents for ABO typing have replaced those prepared from plasma from human donors. MAbs have the advantages of unlimited supply, batch-to-batch consistency, absence of contaminating antibody specificities, and minimal biohazard risk to blood typing staff.
While efforts have been concentrated on making MAbs using the mouse hybridoma system, new techniques keep on developing. In vitro infection of human B-lymphocytes with Epstein-Barr virus (EBV) results in production of transformed, immunoglobulin-secreting lymphoblastoid cells which survive in tissue culture indefinitely and continue to secrete specific antibodies. EBV-transformed human lymphoblastoid cell lines can be fused with a mouse/human myeloma cell line to make MAbs. In this fashion, human anti-D, anti-C and anti-E have been made (Doyle et al. 1985; Crawford et al. 1983). However, the major limitation of this technique is the need for lymphocytes from recently immunized human donors. Thus, this is not an effective method for producing a broad range of blood grouping reagents.
Enriched RBC membrane proteins and synthetic peptides have also been used to immunize animals. Unfortunately, limited success has been obtained due to the inability of the animal to recognize blood group polymorphisms, or because the antigens are only expressed if the protein is embedded in the RBC membrane.
While monoclonal technology has advanced substantially and concerted efforts have been made in many institutions, it has not yet proven possible to make MAb reagents with certain specificities, notably anti-Fy.sup.a and anti-Fy.sup.b. In addition, as human source material, e.g., antibodies of sufficient potency, becomes harder and harder to obtain, it is inevitable that MAb reagents will be needed to replace polyclonal reagents. It is obvious that new approaches are needed if MAbs with specificities that are not currently available are to be made.
In view of the above considerations, it is clear that existing methods for making antibodies are limited. Moreover, it is evident that existing blood typing reagents based on antibodies are limited in both quality and quantity, necessitating new sources of such reagents.
Accordingly, it is one of the purposes of this invention to overcome the above limitations in the manufacture of antibodies, by providing a method which enables designed production of antibodies, particularly monoclonal antibodies to have particular pre-defined specificities. The availability of such designer antibodies thereby enables the manufacture and production of reagents and methods of detecting expression specific proteins which are presently either difficult or even impossible to identify by conventional methods. Therefore, it is another purpose of the invention to provide a method of making a wide range of MAbs capable of use for typing blood samples, investigating functions of proteins, and developing therapeutic reagents.